Fat-storing cells (perisinusoidal stellate cells) were isolated by enzymatic digestion of rat liver and purified by a single-step Nycodenz gradient to yield 11.4 X 10(6) cells per liver, with a purity of 74% and a viability of 76%. Monolayer cultures of fat-storing cells incorporated both [35S]sulfate and [3H]glucosamine into glycosaminoglycans; the rate of incorporation increased with culture time (3-fold between the third and eighth days in culture). About 80% of newly formed glycosaminoglycans were secreted into the medium. Analysis of the types of glycosaminoglycans revealed a different pattern for cells and medium, respectively, which is subject to culture time. Heparan sulfate remains primarily cell-bound and, therefore, has a low fractional secretion rate. Chondroitin sulfate and even more dermatan sulfate are the main types of glycosaminoglycans in the medium. Dermatan sulfate represents about 60% of total medium glycosaminoglycans. In advanced cultures (eighth day), this type becomes the predominant one in the cell layer. The reduction of the molecular weight of native medium-sulfated molecules by papain digestion and beta-elimination and the puromycin-induced inhibition of their synthesis by more than 75% suggest the formation of glycosaminoglycans as complex proteoglycans. It is concluded that fat-storing cells are a major cellular source of dermatan sulfate and chondroitin sulfate in liver connective tissue. Since the pattern of proteoglycans of fat-storing cells closely resembles that found in the fibrotic liver matrix, this cell type might be of pathogenetic significance for the accumulation of chondroitin sulfate and dermatan sulfate in cirrhotic connective tissue.